The proposed studies will examine details of the spontaneous activation of prothrombin and factor X in blood by assaying for thrombin and factor X. Our specific objectives are: 1) To determine the role of factor V in prothrombin as it occurs in blood clotting and the effect of activation of factor V by thrombin on subsequent thrombin formation. When in the clotting sequence factor V is activated, and how this influences the rate and extent of thrombin generation will be studied. These experiments will be based on the determination of thrombin generation by radioimmunoassay in blood from: a) normal controls following various perturbations, e.g., addition of purified thrombin-activated factor V or by inhibition of factor V coagulent activity with specific antibodies and b) factor V deficient patients. Molecular changes which result from thrombin-activation of factor V will be examined immunochemically with antibodies directed against native and activated factor V. 2) To develop a radioimmunoelectrophoretic assay for human factor Xa. With this technique we will study the spontaneous activation of factor X during blood clotting. Also, we will determine the temporal and quantitative inter-relationships between activation of factor X and prothrombin activation. 3) To study the kinetics of inactivation of spontaneously generated thrombin by antithrombin III in blood by quantitating the formation of the thrombin-antithrombin III complex. This will be accomplished by radioimmunoelectrophoresis. Determination of the temporal relationships between thrombin inactivation and blood clotting should provide evidence for the physiological significance of thrombin inhibition by this globulin. 4) To determine whether thrombin and/or factor Xa circulate in a variety of thrombotic diseases and the effect of anticoagulation on the concentration of these factors.